Letters to Nature

Nature 416, 535-539 (4 April 2002) | doi:10.1038/416535a; Received 7 January 2002; Accepted 11 February 2002

Naturally secreted oligomers of amyloid bold beta protein potently inhibit hippocampal long-term potentiation in vivo

Dominic M. Walsh1, Igor Klyubin2, Julia V. Fadeeva1, William K. Cullen2, Roger Anwyl3, Michael S. Wolfe1, Michael J. Rowan2 & Dennis J. Selkoe1

  1. Department of Neurology, Harvard Medical School and Center for Neurologic Diseases, Brigham and Women's Hospital, Boston, Massachusetts 02115, USA
  2. Department of Pharmacology and Therapeutics, Trinity College, Dublin 2, Ireland
  3. Department of Physiology, Trinity College, Dublin 2, Ireland

Correspondence to: Dennis J. Selkoe1 Correspondence and requests for materials should be addressed to D.J.S. (e-mail: Email: dselkoe@rics.bwh.harvard.edu).

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Although extensive data support a central pathogenic role for amyloid beta protein (Abeta) in Alzheimer's disease1, the amyloid hypothesis remains controversial, in part because a specific neurotoxic species of Abeta and the nature of its effects on synaptic function have not been defined in vivo. Here we report that natural oligomers of human Abeta are formed soon after generation of the peptide within specific intracellular vesicles and are subsequently secreted from the cell. Cerebral microinjection of cell medium containing these oligomers and abundant Abeta monomers but no amyloid fibrils markedly inhibited hippocampal long-term potentiation (LTP) in rats in vivo. Immunodepletion from the medium of all Abeta species completely abrogated this effect. Pretreatment of the medium with insulin-degrading enzyme, which degrades Abeta monomers but not oligomers, did not prevent the inhibition of LTP. Therefore, Abeta oligomers, in the absence of monomers and amyloid fibrils, disrupted synaptic plasticity in vivo at concentrations found in human brain and cerebrospinal fluid. Finally, treatment of cells with gamma-secretase inhibitors prevented oligomer formation at doses that allowed appreciable monomer production, and such medium no longer disrupted LTP, indicating that synaptotoxic Abeta oligomers can be targeted therapeutically.

Fibrillar (but not monomeric) forms of Abeta akin to those present in the amyloid plaques of Alzheimer's disease are neurotoxic in culture2, 3. However, relatively weak correlations between fibrillar plaque density and severity of dementia are found in Alzheimer's diseased brains4, 5, whereas correlations between soluble Abeta levels and the extent of synaptic loss and cognitive impairment are stronger6, 7. SDS-stable Abeta oligomers (of relative molecular masses Mr approx8,000 and approx12,000) have been detected by western blotting in the buffer-soluble fraction of Alzheimer's diseased cortex7. These are strikingly similar to soluble, SDS-stable Abeta oligomers produced by certain cultured cells8, 9, 10. We previously confirmed the latter species as Abeta oligomers by amino-terminal radiosequencing and precipitation with carboxy-terminal-specific antibodies8, 10. The levels of these oligomers are specifically augmented by expressing Alzheimer's disease-causing mutations in amyloid precursor protein (APP) or presenilin that increase Abeta42 production11, supporting their pathological relevance. Importantly, young APP transgenic mice undergo synaptic, electrophysiological and behavioural changes before any amyloid plaque formation12, 13, but the nature of the responsible Abeta species in the brain cannot be specifically defined.

To assess the subcellular origin of naturally occurring Abeta oligomers secreted by cells expressing human APP, we prepared microsomes from 7PA2 Chinese hamster ovary (CHO) cells, which stably express the V717F Alzheimer's disease mutation in APP751 (an APP:isoform 751 amino acids in length)8, 10. A highly sensitive immunoprecipitation/western blot method10 revealed both monomers and oligomers of Abeta in the isolated, washed microsomes, and the Abeta dimers in the microsomes co-migrated with those in the conditioned medium (CM) (Fig. 1a). Previous trypsinization of the intact cells did not alter the amounts of the various Abeta assemblies recovered in the microsomes (Fig. 1a, lanes 7 versus 8), supporting their intracellular origin. The co-migrating dimers and trimers present in CM reacted with antibodies specific to the C termini of Abeta40 and Abeta42 (Fig. 1a, lanes 3 and 4). The oligomeric bands were not altered by pre-treatment with 8 M urea or 50% formic acid (Fig. 1b). Previously, radiosequencing of these monomer, dimer and trimer bands in 7PA2 CM confirmed that they are composed of Abeta8.

Figure 1: SDS-stable oligomers of Abeta are produced intracellularly and secreted.
Figure 1 : SDS-stable oligomers of A|[beta]| are produced intracellularly and secreted. Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, or to obtain a text description, please contact npg@nature.com

a, 7PA2 (lanes 2–6) and CHO- (lane 1) conditioned medium (CM) were immunoprecipitated (IP) with antibodies or preimmune serum (Pre) and western blotted with monoclonal antibody 6E10. Microsomes from either 7PA2 (lanes 7–9) or CHO- (land 10) cells were incubated with (lane 7) or without (lanes 8, 10) trypsin, lysed and precleared with APP C-terminal antiserum, C7 (pellet shown, lane 9). Precleared, lysates were then precipitated with R1280 (lanes 7, 8 and 10). The Abeta trimer in microsomal lysates is obscured by residual APP C-terminal fragment, C99 (compare lanes 7–9 with lanes 2–5). Arrow, monomeric Abeta; double arrow, dimeric Abeta; T, trimer. b, Incubation of the R1282 precipitate of 7PA2 CM in either 8 M urea or 50% formic acid did not alter oligomers. c, Centrifugation of CM at 100,000gtimes3 h did not alter oligomer amounts. Relative molecular masses (Mrtimes1,000) are shown on the right-hand side of gel lanes in a, b, Figs 2, 3c–e and 4d, h.

High resolution image and legend (35K)

Extensive immunoelectron microscopy, which readily detects synthetic Abeta protofibrils in the CM of cultured cells14, revealed no such filamentous assemblies in 7PA2 CM (not shown). Ultracentrifugation of the CM at a force (100,000gtimes3 h) that sediments synthetic Abeta fibrils and protofibrils did not bring down any Abeta assemblies, and the amount of oligomers in the supernatant was unaltered (Fig. 1c).

Next, we fractionated total 7PA2 microsomes on discontinuous iodixanol gradients15 and compared the distribution of the oligomers with those of organelle marker proteins. Abeta immunoreactive bands at Mr 4K, 6K and 8K–9K were principally present in fractions 4–7, which were enriched in Golgi-type vesicles (syntaxin-6-positive) and recycling endosomes (transferrin-receptor-positive) but contained only low levels of endoplasmic reticulum proteins (Grp 78 and calnexin) (Fig. 2 and not shown). The microsomal Abeta species co-migrated with the previously confirmed oligomers in the CM (Fig. 2d) and were unreactive with an APP C-terminal antibody (C7) (Fig. 2d) or preimmune serum (not shown). Immunoprecipitation of the subcellular vesicles with antibodies to the free C termini of Abeta40 (2G3) and Abeta42 (21F12) revealed a faint Abeta species of Mr 12K, a putative trimer, in addition to monomers and dimers (not shown). These were again principally localized to iodixanol gradient fractions 4–7, in which Abeta has previously been shown to be generated15. These data suggest that Abeta oligomerization is initiated soon after the generation of Abeta in discrete intracellular vesicles.

Figure 2: SDS-stable oligomers are formed soon after generation of Abeta.
Figure 2 : SDS-stable oligomers are formed soon after generation of A|[beta]|. Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, or to obtain a text description, please contact npg@nature.com

Microsomes fractionated on discontinuous iodixanol gradients15 were lysed and blotted for Grp78 (a), syntaxin 6 (b) and transferring receptor (c). The remainder of each fraction was precleared with C7-protein A (as in Fig. 1) and then precipitated with R1280, and Abeta species blotted with 6E10 (d). CM, medium from the same cells. C7, aliquot of fraction 4, precipitated with C7. Arrow, monomeric Abeta; double arrow, dimeric Abeta. Data are typical of four fractionations.

High resolution image and legend (29K)

To determine whether these cell-derived oligomers of human Abeta have biological activity in vivo, we examined the effect of 7PA2 CM on hippocampal LTP, a measure of synaptic plasticity that is exquisitely sensitive to disruption by synthetic Abeta16. Long-term potentiation of excitatory synaptic transmission in the CA1 area of anaesthetized rats was induced by high-frequency stimulation (HFS). Microinjection of 7PA2 CM (1.5 microl intracerebroventricular, i.c.v.) completely blocked LTP at 3 h post-stimulation; only a waning potentiation lasting <90 min was elicited (Fig. 3a, g). In contrast, a robust LTP that was stable for over 3 h was induced after identical infusion of CM (1.5 microl) from untransfected CHO- sister cultures (Fig. 3a, g). The LTP block was not related to any decrease in baseline transmission, because 7PA2 CM did not alter synaptic responses (EPSPs) in the absence of HFS (Fig. 3a, g).

Figure 3: SDS-stable oligomers of human Abeta block hippocampal LTP in vivo.
Figure 3 : SDS-stable oligomers of human A|[beta]| block hippocampal LTP in vivo. Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, or to obtain a text description, please contact npg@nature.com

a, 7PA2 CM (blue) but not CHO- CM (black, P<0.001) blocked LTP. Insets show typical EPSPs approx5 min pre- (1) and approx3 h post-HFS (2); calibration bars 5 ms/0.5 mV. b, Immunoprecipitation of 7PA2 CM with an antibody to Abeta (R1282, blue, see c) but not to APPS (B5, green) prevented the block of LTP. c, Blots of R1282 precipitates from CM used in b show that Abeta monomer (arrow) and dimer (double arrow) had been efficiently precipitated; compare Abeta retrieved in 1st precipitate versus in a second precipitation (2nd). d, Blotting of remaining CM of samples in c with 22C11 revealed that the R1282 precipitation did not alter APPS-alpha levels. e, Incubation of 7PA2 CM with IDE caused complete loss of Abeta monomers (single arrow), whereas Abeta dimers (double arrow) and trimers were minimally diminished (lanes 1 versus 2). f, Treatment of 7PA2 CM with IDE (red) did not prevent the block of LTP. g, h, Magnitudes of LTP at 3 h post-HFS (% baseline plusminus s.e.m.); asterisks P < 0.05 compared to CHO- (g) or control solution (h). n, Number of animals used per treatment.

High resolution image and legend (120K)

The CM of CHO- and 7PA2 cells should be indistinguishable except for the presence in the latter of the secretory products of human APP metabolism: APPS, Abeta and p3 (Abeta17–40/42). To determine if Abeta caused the LTP block, we compared the effects of 7PA2 CM before and after immunodepletion with R1282, a high-titre polyclonal Abeta antibody. R1282 immunodepletion completely prevented the block of LTP (Fig. 3b, g). Western blotting confirmed that R1282 efficiently precipitated both Abeta monomers and oligomers; a second immunoprecipitation brought down very little additional protein (Fig. 3c). By enzyme-linked immunosorbent assay (ELISA), the R1282 precipitation decreased total Abeta in CM from 5,475 plusminus 897 to 1,253plusminus24 pg ml-1 (meanplusminuss.d., n=3). APPS was poorly precipitated by R1282 (not shown). Correspondingly, the amounts of APPS remaining in CM before and after R1282 immunoprecipitation were indistinguishable (Fig. 3d). The abrogation of the LTP block by R1282 was not due to a nonspecific effect of the immunodepletion or to an undetected decrease in APPS concentration, because immunodepletion of 7PA2 CM with a polyclonal antibody (B5) to APPS had no effect (Fig. 3b, g). The addition of R1282 to CHO- CM likewise did not affect LTP induction (Fig. 3g).

Because neither the Abeta ELISA nor the immunoprecipitation/western blot assay recognizes p3 (ref. 10), we addressed the possibility that p3 in the 7PA2 CM mediated the observed block of LTP. Synthetic human p3 added to CHO- CM at a concentration (11.5 nM) well in excess of that found in 7PA2CM8 did not affect LTP (Fig. 3h).

Next, we asked whether the block of LTP was due to the abundant Abeta monomers or to the less abundant but invariably present SDS-stable Abeta oligomers. To do so, we took advantage of the specificity of insulin-degrading enzyme (IDE) to selectively degrade and remove Abeta monomers from CM (Fig. 3e)17. 7PA2 CM was divided into three equal aliquots: one was held at 4 °C, another was incubated at 37 °C for 12 h, and the third was incubated with purified IDE at 37 °C for 12 h. The latter incubation caused complete loss of detectable Abeta monomer, while leaving the Abeta oligomers virtually unchanged (Fig. 3e, lane 2). The CM aliquot incubated without IDE underwent only a slight decrease in Abeta monomer (Fig. 3e, lane 3). Both the second and third aliquots still caused a block of LTP at 3 h (Fig. 3f, h) that was indistinguishable from that of the first (untreated) aliquot. That IDE itself blocked LTP was excluded when (1) injection of plain medium spiked with IDE did not alter LTP (Fig. 3h), and (2) infusion of IDE-treated 7PA2 CM from which the His-tagged IDE had been removed by metal affinity chromatography still blocked LTP (Fig. 3h, IDE plus MAC). We conclude that naturally secreted, soluble human Abeta oligomers are specifically responsible for the block of hippocampal LTP by 7PA2 CM.

Because LTP was selectively blocked by Abeta oligomers in the absence of monomers, protofibrils or fibrils, we pharmacologically prevented oligomer formation in living cells while retaining substantial monomer production and assessed whether this would preserve normal LTP. We synthesized two structurally dissimilar italic gamma-secretase inhibitors that dose-dependently inhibited Abeta secretion by 7PA2 cells: DAPM (half-maximal inhibitory concentration, IC50 approx10 nM) and MWIII-20 (IC50 approx10 microM)) (Fig. 4a, b). After treatment of cells for 6 h with 10 microM MWIII-20, the dimer (Mr 8K) was only faintly detectable by immunoprecipitation/western blot, whereas approx60% of the monomer signal remained (Fig. 4c, e). For MWIII-20, the IC50 for monomers by this method was approx30 microM, whereas that for dimers was approx10-fold lower. With the more potent DAPM, the IC50 for monomers was approx0.1 microM, whereas that for dimers was approx20-fold lower. Thus, at 0.1 microM, DAPM decreased monomers, but there was a relatively greater loss of dimers, which were now barely or not detectable (Fig. 4d). The percentage decrease of dimer at each concentration was greater than that of monomer in all experiments, and these two values were significantly different at the higher concentrations (P < 0.005) (Fig. 4e, f, asterisks). On the basis of these results, we treated 7PA2 cells for 6 h with 0.1 microM DAPM, confirmed the marked decrease in oligomer levels in this CM (Fig. 4h) and then microinjected an aliquot (1.5 microl i.c.v.) into rats. High-frequency stimulation produced a robust LTP which was fully maintained for over 180 min (Fig. 4g, red symbols). This LTP was indistinguishable from that observed after injection of CHO- CM spiked with the same or greater concentrations of DAPM (1 microM, 144 plusminus 8%, n=3). To control further for the presence of the inhibitor and the fact that treatment with DAPM reduced Abeta monomer production by approx40%, 7PA2 CM was diluted 1:1 with plain DMEM, and DAPM spiked in (to 0.1 microM) just before injection. In contrast to the results obtained using CM from DAPM-treated cells, injection of this diluted 7PA2 CM spiked with DAPM caused a block of LTP typical of that observed previously (P < 0.05, Fig. 4g, blue symbols).

Figure 4: bold italic gamma-secretase inhibitors block Abeta oligomer formation at doses that allow substantial monomer production.
Figure 4 : |[gamma]|-secretase inhibitors block A|[beta]| oligomer formation at doses that allow substantial monomer production. Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, or to obtain a text description, please contact npg@nature.com

Inhibition of 7PA2 cellular Abeta secretion by MWIII-20 (a) and DAPM (b) was quantified by ELISA. Values (means plusminus s.d.) from three independent experiments were normalized to DMSO controls. Insets show inhibitor structures. cd, 7PA2 cells were treated for 6 h with MWIII-20 or DAPM and Abeta species visualized by immunoprecipitation and western blot. e, f, Densitometric quantification of experiments as in c and d; Abeta monomer (black bars) and dimer (grey bars) densities normalized to vehicle control. MWIII-20, n=8 and DAPM, n=5. Asterisk indicates that at a given concentration, decrease in dimer is significantly greater (P < 0.005) than decrease in monomer. g, CM from cells treated with 0.1 microM DAPM (red, n=5) allowed a robust LTP, whereas media from untreated 7PA2 cells (blue, n=4) blocked LTP (insets as in Fig. 3). h, 7PA2 cells were conditioned in plain DMEM with or without DAPM for 6 h; CM was examined by immunoprecipitation and western blot and used for electrophysiology.

High resolution image and legend (100K)

These experiments allow us to attribute an inhibition of hippocampal LTP in vivo specifically to oligomers, not monomers or fibrils, of naturally secreted human Abeta. The ability to collect heterogeneous human Abeta species produced naturally by living cells, combined with the property of IDE to degrade Abeta monomers but not oligomers, enabled us to specify that SDS-stable oligomers of Abeta consistently block hippocampal LTP. In addition to the advantages of this approach over treatments using synthetic Abeta, it provides specificity about the form of Abeta responsible for biological activity that is not achievable in APP transgenic mouse brains. The work demonstrates for the first time that a biochemically defined oligomeric assembly of naturally secreted human Abeta alters hippocampal synaptic efficacy at physiological levels. Our findings strongly support the emerging hypothesis that soluble Abeta oligomers are the principal effectors of the synaptic dysfunction and loss that characterize Alzheimer's disease7, 12, 13, 14, 18, 19, 20. Importantly, the CM we microinjected contained total human Abeta concentrations (mean plusminus s.d.: 3,223 plusminus 1,217 pg ml-1; n=9) very similar to those we previously measured in normal human cerebrospinal fluid (4,003plusminus1,873 pg ml-1, n=32)10.

We provide evidence that after their genesis in intracellular vesicles, Abeta monomers form dimers, trimers and perhaps higher oligomers (Figs 1 and 2), that at least a portion of these oligomers is highly stable, and that some are subsequently secreted (Fig. 1b). The secreted oligomers can interact with neurons in vivo, altering their normal physiology (Fig. 3). Analogous effects may underlie the subtle synaptic changes and impairment of learning and memory documented in young APP transgenic mice12, 13, 20, 21 and in early Alzheimer's disease patients themselves. Our findings do not rule out an additional role for subsequent morphological lesions that characterize Alzheimer's disease and certain transgenic mouse models22.

Synthetic Abeta oligomers block rat hippocampal LTP18 but involve substantially higher doses of single synthetic Abeta species. In transgenic mice overexpressing human APP, some studies report failure of LTP maintenance in 4–5 month old23, 5–7 month old24 or 16 month old25 animals, whereas others find impaired synaptic transmission but no change in LTP at 8–10 months12 or 12–18 months26. Such studies of transgenic mice lack an advantage of our paradigm, namely the ability to study the effects of biochemically defined assembly forms of naturally produced human Abeta at physiological levels, in the absence of any confounding effects of APP overexpression.

An attractive therapeutic approach to Alzheimer's disease would be to reduce selectively the levels of potentially synaptotoxic Abeta oligomers. We show that two chemically distinct, cell-penetrant italic gamma-secretase inhibitors block Abeta dimer and trimer formation at doses which allow appreciable monomer production. In contrast, anti-aggregation compounds would need to be both cell-penetrant and capable of blocking very early Abeta oligomerization; if such molecules did not prevent initial dimerization, they might enhance the levels of potentially synaptotoxic species. Thus, Abeta-lowering compounds (for example, beta- or italic gamma-secretase inhibitors) that prevent intra- and extracellular oligomerization while still allowing significant monomer production would be particularly desirable. Because stable oligomers can potentially arise from a large variety of proteins, both those already implicated in disease27 and those that are not28, the prevention of oligomer formation by reducing monomer concentrations could have wide relevance to the treatment of protein-folding disorders.

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Methods

APP-expressing cells

Chinese hamster ovary cells stably transfected with a complementary DNA coding for APP751 containing the Val717Phe familial Alzheimer's disease mutation (referred to as 7PA2 cells) were cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum as described8, 10.

Immunoprecipitation/western blot analysis

Abeta monomers and oligomers were visualized using a highly sensitive immunoprecipitation/western blot protocol that can readily detect as little as 200 pg of naturally secreted human Abeta10. For quantification of band intensity, appropriate film exposures were scanned and the density of bands determined with AlphaEase software (AlphaInnotech).

Subcellular fractionation

Total cellular microsomes were prepared from thirty 10-cm dishes of 7PA2 cells (approx3times108 cells), and fractionated on iodixanol step gradients essentially as described previously15. The resulting gradient was fractionated into 1-ml aliquots and a 100timesstock of protease inhibitors and 10% NP40 was added to each fraction to yield 1timesprotease inhibitors and 1% NP40. Fractions were vortexed, and aliquots (50 microl) removed for western blotting15. After clearing with a C7-protein A conjugate, samples were examined by immunoprecipitation/western blot10.

Abeta ELISA

Enzyme-linked immunosorbent assay (ELISA) for Abeta1-total was performed as before10. Full-length APP and APPS-alpha were not detected by either assay.

Expression and purification of IDE

Recombinant human IDE (N-terminally tagged with both polyhistidine and haemagglutinin) was expressed in BL21(DE3) Escherichia coli cells and purified using the TALON metal affinity kit (Clontech Laboratories), as described previously29. The specificity of the purified IDE was confirmed by the degradation of 125I-Abeta and 125I-insulin and by inhibition with cold insulin or 1,10-phenanthroline17.

Preparation of conditioned medium for microinjection and electrophysiology

7PA2 and untransfected CHO- cells were grown to near confluency and allowed to condition plain DMEM for approx16 h. Conditioned medium was cleared of cells (200gtimes10 min, 4 °C) and used directly for electrophysiology or else treated to remove Abeta monomers or both Abeta monomers and oligomers. To fully deplete monomers but retain oligomers, CM (6 ml) was incubated with recombinant IDE (475 microg) for 12 h at 37 °C. In some experiments, the His-tagged IDE was then removed by metal affinity chromatography29.

Electrophysiology

Experiments were carried out on urethane anaesthetized male adult Wistar rats. Single-pathway recordings of field excitatory postsynaptic potentials (EPSPs) were made from the stratum radiatum in the CA1 area of the hippocampus in response to stimulation of the ipsilateral Schaffer collateral/commissural pathways as described previously16. Test EPSPs were evoked at a frequency of 0.033 Hz and at a stimulation intensity adjusted to give an EPSP amplitude of 50% of maximum. The HFS protocol for inducing LTP consisted of 10 trains of 20 stimuli, inter-stimulus interval 5 ms (200 Hz), inter-train interval 2 s. The intensity was increased to give an EPSP of 75% of maximum amplitude during the HFS. To inject samples a cannula was implanted in the lateral cerebral ventricle (coordinates: 0.5 mm anterior to bregma and 1.0 mm right of midline) just before electrode implantation16. Conditioned medium samples (1.5 microl) were injected over a 2 min period, 10 min before HFS. Control injections comprised superQ water or 0.9% NaCl. LTP was measured as the mean plusminus s.e.m. per cent of the baseline field EPSP amplitude recorded over at least a 30-min baseline period. Similar results were obtained when the EPSP slope was measured. Statistical comparisons used paired and unpaired Student's t-tests.

Synthesis of bold italic gamma-secretase inhibitors

The general method of ref. 30 was followed to make MWIII-20, which is carbamic acid, [3-[(L-leucyl-L-leucyl-O-methyl ester)carbonyl](phenylmethyl)amino]-2R-hydroxy-1S-(phenylmethyl)propyl]-1,1-dimethylethyl ester). DAPM (N-[N-3,5-difluorophenacetyl)-L-alanyl[-S-phenylglycine methyl ester) has been reported in patent applications WO9822441-A2 and WO9822494-A2 filed by Athena Neurosciences and Eli Lilly. The identity of each compound was confirmed by 1H-NMR and mass spectrometry.

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References

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Acknowledgements

We thank M. Rosner and V. Chesneau for the gift of the pProExH6HA IDE expression vector, B. Zheng for ELISA analysis, S. Mansourian for assistance in the preparation of illustrations and W. T. Kimberly, W. P. Esler and D. M. Hartley for discussions. Supported by NIH grants (to D.J.S. and M.S.W.) and by Enterprise Ireland and the Health Research Board Ireland (M.R. and R.A.).

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Competing interests statement

The authors declare no competing financial interests.